[N,N,N',N',N'',N''-hexaethyl-29H,31H-phthalocyaninetrimethylaminato(2-)-N29,N30,N31,N32]copper

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2012-2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP and OECD testing guideline compliant study with well-characterized test material. Based on ECHA-guidance, a default reliability of 2 is assigned to account for read-across.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, Horst, The Netherlands
- Age at study initiation: (P) 11-12 weeks at the start of the treatment period
- Weight at study initiation: the initial body weight variation did not exceed ±20% of the mean weight for each sex
- Housing: Macrolon cages (4 rats/sex)
- Diet: Cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Witham,England), ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2°C with a maximum of 24.9 °C
- Humidity (%): 44.2% and not exceeding 65%
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

IN LIFE DATES:
From 2012-05-01 to 2012-06-15

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% in water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dilutions of the test substance in the vehicle and the vehicle for dosing the controls were prepared weekly and stored in a refrigerator (2-10ºC), in portions sufficient for one day. During dosing the dose formulation was stirred continuously.

VEHICLE
- Concentration in vehicle: The vehicle was 1% aqueous CMC, dosing volume was 15 mL/kg bw/day.

Details on mating procedure:

- M/F ratio per cage: During the premating period, the animals were housed in groups of 4rats/sex. For mating, one male and one female were housed together (1:1). Mated females were housed individually.
- Length of cohabitation: Animals were caged together until mating occurred or 1 week had elapsed.
- Proof of pregnancy: vaginal smears, the day on which sperm was detected in the vaginal smear was considered as gestation day 0.
- After successful mating each pregnant female was caged: Females were caged individually for the birth and rearing of their pups.
- Dams were allowed to raise their litter until sacrifice on day 4 of lactation or shortly thereafter.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogenicity and concentration of the test item in the dose formulations was assessed twice during the treatment period.

Duration of treatment / exposure:

ADMINISTRATION OF THE TEST ITEM
The test item was administered to the animals daily by oral gavage for 46 days. A dosing volume of 15 mL/kg body weight was used for all groups. Dosing volume was adjusted based on the latest body weight. For the females during gestation the dosing volume remained constant from gestation day 14 up to the end of gestation. Male animals were dosed during a 2-week premating period, and during mating and up to the day of sacrifice. The female animals were dosed during a 2-week premating period, and during mating, gestation and lactation up to the day of sacrifice (approx. day 4 of lactation).

Frequency of treatment:

- once daily (on a single occasion, animals 15 and 21 of the control group and animals 37, 41, 43 and 47 in the low dose group were erroneously dosed twice)

Details on study schedule:

Age at mating of the mated animals in the study: young adult

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12 rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 2-weeks dose range finding study with the test item in rats.

Positive control:

None

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily in the morning, all cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study.

DETAILED CLINICAL OBSERVATIONS: Yes; time schedule : prior to the first exposure and at the end of the study as part of the functional observation battery (FOB)

NEURO-BEHAVIOURAL TESTING (FOB) AND SPONTANEOUS MOTOR ACTIVITY
During neuro-behavioural testing, the observer was unaware of the treatment of the animals. FOB and spontaneous motor activity were assessed in all study animals during the predose phase and in 5 animals/sex/group at the end of the study.

BODY WEIGHT: Yes
- Time schedule for examinations: shortly before the start of administration of the test item and at the start of the study (day 0). Males were weighed weekly until sacrifice. Females were weighed once per week during the premating period. Mated females were weighed on days 0, 7, 14 and 21 during presumed gestation and on day 0 and 4 of lactation. Non-mated females were weighed once per week after the mating period. In addition, the animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios. For female 1 in the control group no copulation date was registered and therefore body weight for this female was recorded once weekly, but not on gestation days 0, 7, 14 and 21.

FOOD CONSUMPTION:
Feed consumption was measured per cage over weekly intervals during the study, with exemption of the mating period, during which no feed consumption was registered.

HAEMATOLOGY
Prior to sacrifice, 5 animals/sex/group were fasted overnight (water was freely available) and blood was taken from the aorta during necropsy, whilst under CO2/O2 anaesthesia. K3-EDTA was used as anticoagulant. In each sample the following determinations were carried out:
haemoglobin
packed cell volume
red blood cell count
reticulocytes
total white blood cell count
differential white blood cell counts (neutrophils, lymphocytes, eosinophils,
basophils, monocytes)
prothrombin time
thrombocyte count
mean corpuscular volume (MCV; calculated)
mean corpuscular haemoglobin (MCH; calculated)
mean corpuscular haemoglobin concentration (MCHC; calculated)
activated partial prothoplastin time (APTT)
red blood cell districution width (RDW)

CLINICAL CHEMISTRY
Prior to sacrifice, 5 animals/sex/group were fasted overnight (water was freely available) and blood was taken from the aorta during necropsy, whilst CO2/O2 anaesthesia. Blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. The following measurements were made in the plasma:
alkaline phosphatase activity (ALP),
aspartate aminotransferase activity (ASAT),
alanine aminotransferase activity (ALAT),
gamma glutamyl transferase activity (GGT),
total protein,
albumin,
ratio albumin to globulin (calculated),
urea,
creatinineme,
glucose (fasting),
bilirubin (total),
cholesterol (total),
triglycerides,
phospholipids,
calcium (Ca),
sodium (Na),
potassium (K),
chloride (Cl),
inorganic phosphate (PO4),
bile acids.

OTHER:
PARTURITION
At the end of the gestation period (GD 21), females were examined twice daily for signs of parturition. Any difficulties occurring during parturition were recorded.

Litter observations:

PARAMETERS EXAMINED
The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups and grossly malformed pups were evaluated on days 0 and 4 of lactation. The pups were individually weighed on days 0 and 4 of lactation. Mean pup weight was calculated per sex and for both sexes combined.

GROSS EXAMINATION OF DEAD PUPS:
A necropsy was performed on stillborn pups and pups that died during the study. All macroscopic abnormalities were recorded. To keep nest disturbance to a minimum the litters were examined only once daily for dead pups.

Postmortem examinations (parental animals):

BLOOD SAMPLING FOR HORMONE ANALYSIS
During necropsy blood was taken from the aorta under CO2/O2 anaesthesia from 5 overnight fasted animals/sex/group for potential future determination of TSH, T3 and T4 hormone levels. Samples were stored as serum at -18°C.

GROSS NECROPSY AND HISTOLOGY OF PARENTAL ANIMALS
All surviving male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia at necropsy and then examined grossly for pathological changes. Male animals were sacrificed after the mating period. Female animals were sacrificed at or shortly after day 4 of lactation. A necropsy was performed on animals that died intercurrently (if not precluded by autolysis) or that had to be killed because they were moribund. Prior to preservation of organs/tissues, the following organ weights were recorded: adrenals, brain, heart, kidneys, liver, ovaries, prostate, seminal vesicles, spleen, thymus, thyroid, uterus. Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes which were preserved in Bouin's fixative:

- ovaries (after counting the corpora lutea)
- uterus (after counting of the implantation sites)
- testes
- epididymides
- seminal vesicles
- prostate
- all gross lesions
In addition of 5 animals/sex/group following organs were preserved:
adrenals
bone marrow (femur)
brain (including sections of cerebrum, cerebellum, medulla/pons)
caecum
cervix
clitorial gland
colon
coagulation gland
duodenum
eyes
femur including joint
heart
ileum
jejunum (including Peyer's patches)
kidneys
liver
lungs
mesenterial and axillary lymph nodes
ovaries
peripheral nerve (sciatic or tibial)
pituitary gland
preputial gland
prostate
rectum
seminal vesicles (including coagulation gland)
skeletal muscle (thigh)
spinal cord (cervical, mid-thoracic and lumbar)
spleen
stomach*
thymus
thyroid (including parathyroid)
trachea
urinary bladder
uterus
vagina
* Non glandular (“forestomach”) and glandular (fundus, pylorus) parts were examined
microscopically.

Microscopic examination was performed on the collected organs of all animals of the control (group 1) and high dose group (group 4). If treatment-related changes were observed in the high-dose group, the evaluation of these tissues/organs was extended to the intermediate-dose groups (2 and 3).
In addition, reproductive organs of males that failed to sire (did not mate or mated females were not pregnant) and females that were non-mated or non-pregnant, of the low- and mid-dose groups, were microscopically examined. Furthermore, organs showing gross lesions of animals of all groups were microscopically examined.

FERTILITY AND REPRODUCTIVE PERFORMANCE
For each mating the following data were presented for each group:
- number of females placed with males
- number of males mated with females
- number of successful copulations (= number of females mated)
- number of males that became sire
- number of pregnant females as demonstrated by the presence of implantation
sites observed at necropsy
- number of females surviving delivery
- number of females with liveborn and (all) stillborn pups
- number of pups delivered (live- and stillborn)
- number of live pups at day 1 and 4
- number of pups lost
- number of litters lost entirely
- number of male pups at day 1 and 4
- number of corpora lutea
- number of implantation sites
- number of lost implantations
- litter size

Postmortem examinations (offspring):

SACRIFICE
At necropsy of the dams, at or shortly after day 4 of lactation, pups were killed by appropriate techniques.

PATHOLOGY OF PUPS
Grossly malformed pups were sacrificed and examined. All pups were examined externally at the necropsy of the dams. The pups were killed and and stored in a freezer for possible skilled analyses.

Statistics:

The resulting data were analyzed using the methods mentioned below. Other statistical tests were performed when considered appropriate. P < 0.05 was considered as a level of significance.
- Body weight, body weight gain, food consumption and organ weights data were subjected to one way analysis of variance (ANOVA), followed by Dunnett or Kruskal-Wallis analysis.
- Fisher's exact probability test or the Chi-square test followed by Fisher were used to evaluate the number of mated and pregnant females, the number of pregnant females with implants but no pups, females with live pups, females with stillborn pups, live and dead fetuses or pups and the numbers of litters lost entirely.
- Pre-coital time (mean number of days), the duration of gestation, the number of corpora lutea and implantation sites, the total number of pups delivered (mean), the mean number of live pups per litter and pre- and post-implantation loss (%) were evaluated by Kruskal- Wallis nonparametric analysis of variance.
- Haematology and clinical chemistry parameters were subjected to one way ANOVA followed by Dunnett’s multiple comparison tests.
- Mortality data and data of the pathology of parent animals were evaluated by the Fisher’s exact probability test.

Reproductive indices:

Male and female fertility indices, mating index, female fecundity index, gestation index

Offspring viability indices:

live birth index, viability index day 1-4 (sex ratio), pup mortality day 1 or 4

MORTALITY AND CLINICAL SIGNS
There was no test item related mortality. One male of the low dose group (male animal 44) was found dead and partly cannibalized on day 17 of dosing, during the mating period. The animal showed no clinical signs leading to its death. Histopathology revealed widespread blue particles (test item) in the lungs confirming faulty dosing of the animal. Male animal 34 in the low dose group revealed hunched posture during the study. At autopsy an abscess in the thorax was found, confirming faulty dosing of the animal. No treatment related clinical signs were observed in males during the premating, mating and postmating period. No treatment related clinical signs were observed in females during the premating, mating, gestation and lactation period.
In several animals the tip of tail was missing or showed encrustations. Upon healing of the wound, the tail was considered complete and no observations were recorded. Male animals 40 (low dose) and 80 (high dose) showed grunting on several occasions.

BODY WEIGHT AND BODY WEIGHT CHANGE
No effects on body weight or body weight change were observed in males and females throughout the study.

FOOD CONSUMPTION
No effect on food consumption was observed in males and females throughout the study. In the post-mating period a decrease in mean food consumption (mg/kg bw/day) was observed in the mid and high dose group males. However, as this was not observed in the food consumption when expressed as mg/animal/day, this was not considered to be a treatment-related effect.

HAEMATOLOGY
There were no effects on haematology parameters.

CLINICAL CHEMISTRY
There were no effects on clinical chemistry parameters.

NEUROBEHAVIOURAL TESTING (FOB AND MOTOR ACTIVITY)
The results of the neurobehavioural observations and motor activity did not indicate any neurotoxic potential of CAS no. 75247-18-6 in rats.

FERTILITY AND REPRODUCTIVE PERFORMANCE
In each dosing group 12 females were placed with 12 males, resulting in 9/12 mated females in the control group, 11/12 mated females in the low and mid dose groups and 12/12 mated females in the high dose group. One female in the control group (female 1) seemed not mated, but was pregnant and delivered a litter after all. For this female no insemination date has been established. Male and female fertility indices and the mating index were slightly lower in the control group. The mean number of mating days until insemination was comparable in all groups. The mean number of corpora lutea and the mean number of implantation sites was comparable in all groups. The mean pre-implantation loss showed large inter group variations, but no treatment related effects were observed. Mean prenatal loss was comparable in all groups. All females survived delivery, resulting in 9/12, 10/12, 11/12 and 12/12 litters in the control group, low dose, mid dose and high dose groups, respectively. All females delivered litters with liveborn pups. One litter in the low dose group comprised one stillborn pup amongst liveborn pups. There were no litters comprising stillborn pups only.

ORGAN WEIGHTS OF PARENTAL ANIMALS
No effects on organ weights were observed in male and female animals.

MACROSCOPIC EXAMINATION OF PARENTAL ANIMALS
At necropsy many animals treated with the test substance showed blue (or bluish/green) discoloration of the (contents of the) gastro-intestinal tract and in a few cases also at mesenteric lymph nodes. This was obviously due to the color of the test substance. One male of the low dose group, no 34, had a green nodule (2 cm) in the thoracic cavity, which appeared to be an abscess. This was clearly caused by faulty dosing (which means that the gavage needle accidently had penetrated the esophagus wall), resulting in a local abscess with blue coloured particles present in the interstitial tissue. In one female of the low-dose group, no 45, the vaginal aperture was missing, which accounted for the dilated uterus and vagina and the observation that this animal had not mated. The other gross changes were unremarkable.

MICROSCOPIC EXAMINATION OF PARENTAL ANIMALS
As a standard procedure at necropsy the intestines were flushed with formalin to enable proper preservation of the mucosa. Consequently the blue discoloured contents were flushed out for a large part and therefore not present in the slides. In several animals some intraluminal blue content was still visible in slides of the intestines. It was noted at the microscopic evaluation, but was omitted from the summarizing histopathology table because of its limited relevance. There was no clear evidence that the blue substance was able to pass the intestinal wall, because it was only found in the lumen of the intestinal tract but not in other organs or tissues. There were a few exceptions though: there were two cases of faulty dosing in the low dose group (animals 34 (see above) and 44 (with widespread blue particles in the lungs; this animal was found dead on day 17 of the study) and in the high dose group (animal 84 which had some blue particles in the surrounding adipose tissue of the mesenteric lymph nodes which could not be explained). Further, microscopic examination of the sampled organs and tissues did not reveal treatment related histopathological changes. The histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They were common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to treatment.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproductive effects were noted.

LITTER SIZE AND SEX
The mean number of pups delivered per litter was comparable in all groups (12.6, 11.6, 13.3 and 11.8 in the control, low dose, mid dose and high dose groups, respectively). The mean percentage male littermates at delivery was comparable in all groups (52.2%, 50.4%, 49.3% and 49.6% in the control, low dose, mid dose and high dose groups, respectively).

PUP OBSERVATIONS
Five female pups from one litter in the mid dose group were found to be cold and another female pup of this litter showed paralyzed legs. No treatment related clinical signs were observed during lactation.

PUP BODY WEIGHT
Mean pup body weight was comparable for males and females in all groups on days 0 and 4 of lactation.

VIABILITY INDEX
A total number of 113, 115, 146 and 141 pups were liveborn in the control, low dose, mid dose and high dose groups, respectively. One pup in the low dose group was stillborn and a total of 4, 3 and 9 pups in the control, low dose and mid dose group were missing and had died on lactation day 4. No pups in the high dose were lost during day 0 and 4 of lactation. There were no litters showing complete litter loss. The viability indices on day 4 of lactation (96.5%, 97.4%, 93.8% and 100% for the control, low dose, mid dose and high dose groups, respectively) were comparable in all groups.

MACROSCOPIC OBSERVATIONS OF STILLBORN PUPS OR PUPS THAT DIED DURING LACTATION
Macroscopic examination of the stillborn pup in the low dose group (31-06) showed that the pup had no milk in the stomach.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No developmental effects were noted.

Reproductive effects observed:

not specified

A homogeneous distribution of the test item in the dose formulation was ensured by means of continuous stirring prior to dosing. The achieved concentrations were generally considered acceptable.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (422) (read across)

In a reliable OECD TG 422 / GLP study (TNO, 2012), 12 male and 12 female Wistar rats received the test item, CAS number 75247 -18 -6, by daily oral (gavage). Male animals were dosed during the premating period (2 week), and during mating (1 week) and up to the day of sacrifice. Female animals were dosed during the premating period, and during mating, gestation and lactation up to the day of sacrifice (approximately day 4 of lactation) for a period of 46 days. The dose-levels were 0,100, 300 and 1000 mg/kg bw/day. The study included check of clinical signs and mortality, body weight and food consumption, investigation of Functional Observation Battery (FOB) endpoints and motor activity, blood and urine analysis, macroscopic and microscopic examination, assessment of fertility and reproductive performance and observation and examination of pups. Two animals died due to faulty dosing. No effects on body weight/body weight changes, organ weight and food consumption were observed throughout the study. There were no substance-induced effects on the male and female reproductive performance, nor on the progeny of the parental rats at any dose-level. In one female of the low-dose group the vaginal aperture was missing. The other gross changes were unremarkable. Microscopic examination of parental animals revealed no treatment related effects. The mean number of pups delivered per litter was comparable in all groups. Five female pups from one litter in the mid dose group were found to be cold and another female pup of this litter showed paralyzed legs. No treatment related clinical signs were observed during lactation. Mean pup body weight was comparable for males and females in all groups on days 0 and 4 of lactation. Macroscopic examination of one stillborn pup in the low dose group showed that the pup had no milk in the stomach. A total number of 113, 115, 146 and 141 pups were liveborn in the control, low dose, mid dose and high dose group, respectively. There were no litters showing complete litter loss. The viability indices on lactation day 4 were comparable in all groups. On the basis of these results, the NOAEL (reproduction/development) was considered to be 1000 mg/kg bw/day in males and females.

Short description of key information:
Based on an OECD Guideline 422 and GLP study with CAS number 75247-18-6 (read across approach, please refer to chapter 13 of the IUCLID, Assessment reports), the NOAEL for reproductive toxicity was considered to be 1000 mg/kg bw/day for all relevant endpoints, which was the highest dose tested.

Justification for selection of Effect on fertility via oral route:
The surrogate CAS number 75247-18-6 is used for a read-across to the target substance CAS number 28654-73-1.

Effects on developmental toxicity

Description of key information

In an OECD Guideline 422 and GLP study with CAS number 75247-18-6 (read across approach, please refer to chapter 13 Assessment reports) in rats, the NOAEL for developmental toxicity was at the highest test dose of 1000 mg/kg bw/day. 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP and OECD testing guideline compliant study with well characterized test material. A default reliability of 2 is assigned to account for read-across.

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, Horst, The Netherlands
- Age at study initiation: (P) 11-12 weeks at the start of the treatment period
- Weight at study initiation: the initial body weight variation did not exceed ±20% of the mean weight for each sex
- Housing: Macrolon cages (4 rats/sex)
- Diet: Cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Witham,England), ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2°C with a maximum of 24.9 °C
- Humidity (%): 44.2% and not exceeding 65%
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

IN LIFE DATES:
From 2012-05-01 to 2012-06-15

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dilutions of the test substance in the vehicle and the vehicle for dosing the controls were prepared weekly and stored in a refrigerator (2-10ºC), in portions sufficient for one day. During dosing the dose formulation was stirred continuously.

VEHICLE
- Concentration in vehicle: The vehicle was 1% aqueous CMC, dosing volume was 15 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogenicity and concentration of the test item in the dose formulations was assessed twice during the treatment period.

Details on mating procedure:

- M/F ratio per cage: During the premating period, the animals were housed in groups of 4 rats/sex. For mating, one male and one female were housed together (1:1). Mated females were housed individually.
- Length of cohabitation: Animals were caged together until mating occurred or 1 week had elapsed
- Proof of pregnancy: vaginal smears, the day on which sperm was detected in the vaginal smear was considered as gestation day 0.
- After successful mating each pregnant female was caged: Females were caged individually for the birth and rearing of their pups.
- Dams were allowed to raise their litter until sacrifice on day 4 of lactation or shortly thereafter.

Duration of treatment / exposure:

ADMINISTRATION OF THE TEST ITEM
The test item was administered to the animals daily by oral gavage for 46 days. A dosing volume of 15 mL/kg bw was used for all groups. Dosing volume was adjusted based on the latest body weight. For the females during gestation the dosing volume remained constant from gestation day 14 up to the end of gestation. Male animals were dosed during a 2-week premating period, and during mating and up to the day of sacrifice. The female animals were dosed during a 2-week premating period, and during mating, gestation and lactation up to the day of sacrifice (approx. day 4 of lactation).

Frequency of treatment:

- once daily (on a single occasion, animals 15 and 21 of the control group and animals 37, 41, 43 and 47 in the low dose group were erroneously dosed twice)

Duration of test:

From premating to day 4 of lactation (46 days)

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12 rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were based on the results of a 2-weeks dose range finding study with the test item in rats.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily in the morning, all cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study

DETAILED CLINICAL OBSERVATIONS: Yes; time schedule : prior to the first exposure and at the end of the study as part of the functional observation battery (FOB)

NEURO-BEHAVIOURAL TESTING (FOB) AND SPONTANEOUS MOTOR ACTIVITY
During neuro-behavioural testing, the observer was unaware of the treatment of the animals. FOB and spontaneous motor activity were assessed in all study animals during the predose phase and in 5 animals/sex/group at the end of the study.

BODY WEIGHT: Yes
- Time schedule for examinations: shortly before the start of administration of the test item and at the start of the study (day 0). Males were weighed weekly until sacrifice. Females were weighed once per week during the premating period. Mated females were weighed on days 0, 7, 14 and 21 during presumed gestation and on day 0 and 4 of lactation. Non-mated females were weighed once per week after the mating period. In addition, the animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios. For female 1 in the control group no copulation date was registered and therefore body weight for this female was recorded once weekly, but not on gestation days 0, 7, 14 and 21.

FOOD CONSUMPTION:
Feed consumption was measured per cage over weekly intervals during the study, with exemption of the mating period, during which no feed consumption was registered.

PARTURITION
At the end of the gestation period (GD 21), females were examined twice daily for signs of parturition. Any difficulties occurring during parturition were recorded.

HAEMATOLOGY
Prior to sacrifice, 5 animals/sex/group were fasted overnight (water was freely available) and blood was taken from the aorta during necropsy, whilst under CO2/O2 anaesthesia. K3-EDTA was used as anticoagulant. In each sample the following determinations were carried out:
haemoglobin
packed cell volume
red blood cell count
reticulocytes
total white blood cell count
differential white blood cell counts (neutrophils, lymphocytes, eosinophils,
basophils, monocytes)
prothrombin time
thrombocyte count
mean corpuscular volume (MCV; calculated)
mean corpuscular haemoglobin (MCH; calculated)
mean corpuscular haemoglobin concentration (MCHC; calculated)
activated partial prothoplastin time (APTT)
red blood cell districution width (RDW)

CLINICAL CHEMISTRY
Prior to sacrifice, 5 animals/sex/group were fasted overnight (water was freely available) and blood was taken from the aorta during necropsy, whilst CO2/O2 anaesthesia. Blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. The following measurements were made in the plasma:
alkaline phosphatase activity (ALP),
aspartate aminotransferase activity (ASAT),
alanine aminotransferase activity (ALAT),
gamma glutamyl transferase activity (GGT),
total protein,
albumin,
ratio albumin to globulin (calculated),
urea,
creatinineme,
glucose (fasting),
bilirubin (total),
cholesterol (total),
triglycerides,
phospholipids,
calcium (Ca),
sodium (Na),
potassium (K),
chloride (Cl),
inorganic phosphate (PO4),
bile acids.

BLOOD SAMPLING FOR HORMONE ANALYSIS
During necropsy blood was taken from the aorta under CO2/O2 anaesthesia from 5 overnight fasted animals/sex/group for potential future determination of TSH, T3 and T4 hormone levels. Samples were stored as serum at -18°C.

GROSS NECROPSY AND HISTOLOGY OF PARENTAL ANIMALS
All surviving male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia at necropsy and then examined grossly for pathological changes. Male animals were sacrificed after the mating period. Female animals were sacrificed at or shortly after day 4 of lactation. A necropsy was performed on animals that died intercurrently (if not precluded by autolysis) or that had to be killed because they were moribund. Prior to preservation of organs/tissues, the following organ weights were recorded: adreanals, brain, heart, kidneys, liver, ovaries, prostate, seminal vesicles, spleen, thymus, thyroid, uterus. Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes which were preserved in Bouin's fixative:

- ovaries (after counting the corpora lutea)
- uterus (after counting of the implantation sites)
- testes
- epididymides
- seminal vesicles
- prostate
- all gross lesions
In addition of 5 animals/sex/group following organs were preserved:
adrenals
bone marrow (femur)
brain (including sections of cerebrum, cerebellum, medulla/pons)
caecum
cervix
clitorial gland
colon
coagulation gland
duodenum
eyes
femur including joint
heart
ileum
jejunum (including Peyer's patches)
kidneys
liver
lungs
mesenterial and axillary lymph nodes
ovaries
peripheral nerve (sciatic or tibial)
pituitary gland
preputial gland
prostate
rectum
seminal vesicles (including coagulation gland)
skeletal muscle (thigh)
spinal cord (cervical, mid-thoracic and lumbar)
spleen
stomach*
thymus
thyroid (including parathyroid)
trachea
urinary bladder
uterus
vagina
* Non glandular (“forestomach”) and glandular (fundus, pylorus) parts were examined
microscopically.

Microscopic examination was performed on the collected organs of all animals of the control (group 1) and high dose group (group 4). If treatment-related changes were observed in the high-dose group, the evaluation of these tissues/organs was extended to the intermediate-dose groups (2 and 3).
In addition, reproductive organs of males that failed to sire (did not mate or mated females were not pregnant) and females that were non-mated or non-pregnant, of the low- and mid dose groups, were microscopically examined. Furthermore, organs showing gross lesions of animals of all groups were microscopically examined.

FERTILITY AND REPRODUCTIVE PERFORMANCE
For each mating the following data were presented for each group:
- number of females placed with males
- number of males mated with females
- number of successful copulations (= number of females mated)
- number of males that became sire
- number of pregnant females as demonstrated by the presence of implantation
sites observed at necropsy
- number of females surviving delivery
- number of females with liveborn and (all) stillborn pups
- number of pups delivered (live- and stillborn)
- number of live pups at day 1 and 4
- number of pups lost
- number of litters lost entirely
- number of male pups at day 1 and 4
- number of corpora lutea
- number of implantation sites
- number of lost implantations
- litter size

OVARIES AND UTERINE CONTENT
The ovaries and uterine content was examined after termination: yes
Examinations included:
- gravid uterus weight: No data
- number of corpora lutea: Yes
- number of implantations: Yes
- number of early resorptions: Preimplantation loss was determined
- number of late resorptions: Postimplantation loss and live birth index were determined
- other: see FERTILITY AND REPRODUTIVE PERFORMANCE

Fetal examinations:

SACRIFICE
At necropsy of the dams, at or shortly after day 4 of lactation, pups were killed by appropriate techniques.

PATHOLOGY OF PUPS
Grossly malformed pups were sacrificed and examined. A necropsy was performed on stillborn pups and pups dying during the study; macroscopic abnormalities were recorded.

All pups were examined externally at the necropsy of the dams. The pups were killed and stored in a freezer for possible skeletal analyses.

Statistics:

The resulting data were analyzed using the methods mentioned below. Other statistical tests were performed when considered appropriate. P < 0.05 was considered as a level of significance.
- Body weight, body weight gain, food consumption and organ weights data were subjected to one way analysis of variance (ANOVA), followed by Dunnett or Kruskal-Wallis analysis.
- Fisher's exact probability test or the Chi-square test followed by Fisher were used to evaluate the number of mated and pregnant females, the number of pregnant females with implants but no pups, females with live pups, females with stillborn pups, live and dead fetuses or pups and the numbers of litters lost entirely.
- Pre-coital time (mean number of days), the duration of gestation, the number of corpora lutea and implantation sites, the total number of pups delivered (mean), the mean number of live pups per litter and pre- and post-implantation loss (%) were evaluated by Kruskal- Wallis nonparametric analysis of variance.
- Haematology and clinical chemistry parameters were subjected to one way ANOVA followed by Dunnett’s multiple comparison tests.
- Mortality data and data of the pathology of parent animals were evaluated by the Fisher’s exact probability test.

Indices:

REPRODUCTIVE INDICES
Male and female fertility indices, mating index, female fecundity index, gestation index

OFFSPRING VIABILITY INDICES
live birth index, viability index day 1-4 (sex ratio), pup mortality day 1 or 4

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITY AND CLINICAL SIGNS
There was no test item related mortality. One male of the low dose group (male animal 44) was found dead and partly cannibalized on day 17 of dosing, during the mating period. The animal showed no clinical signs leading to its death. Histopathology revealed widespread blue particles (test item) in the lungs confirming faulty dosing of the animal. Male animal 34 in the low dose group showed hunched posture during the study and at autopsy an abscess in the thorax was found, confirming faulty dosing of the animal. No treatment related clinical signs were observed in male during the premating, mating and postmating period. No treatment related clinical signs were observed in females during the premating, mating, gestation and lactation period.
In several animals the tip of tail was missing or showed encrustations. Upon healing of the wound, the tail was considered complete and no observations were recorded. Male animals 40 (low dose) and 80 (high dose) showed grunting on several occasions.

BODY WEIGHT AND BODY WEIGHT CHANGE
No effects on body weight or body weight change were observed in males and females throughout the study.

FOOD CONSUMPTION
No effect on food consumption was observed in males and females throughout the study. In the post-mating period a decrease in mean food consumption (mg/kg bw/day) was observed in the mid and high dose group males. However, as this was not observed in the food consumption when expressed as mg/animal/day, this was not considered to be a treatment-related effect.

HAEMATOLOGY
There were no effects on haematology parameters.

CLINICAL CHEMISTRY
There were no effects on clinical chemistry parameters.

NEUROBEHAVIOURAL TESTING (FOB AND MOTOR ACTIVITY)
The results of the neurobehavioural observations and motor activity did not indicate any neurotoxic potential of CAS no. 28654-73-1 in rats.

FERTILITY AND REPRODUCTIVE PERFORMANCE
In each dosing group 12 females were placed with 12 males, resulting in 9/12 mated females in the control group, 11/12 mated females in the low and mid dose groups and 12/12 mated females in the high dose group. One female in the control group (female 1) seemed not mated, but was pregnant and delivered a litter after all. For this female no insemination date has been established. Male and female fertility indices and the mating index were slightly lower in the control group. The mean number of mating days until insemination was comparable in all groups. The mean number of corpora lutea and the mean number of implantation sites was comparable in all groups. The mean pre-implantation loss showed large inter group variations, but no treatment related effects were observed. Mean prenatal loss was comparable in all groups. All females survived delivery, resulting in 9/12, 10/12, 11/12 and 12/12 litters in the control group, low dose, mid dose and high dose groups, respectively. All females delivered litters with liveborn pups. One litter in the low dose group comprised one stillborn pup amongst liveborn pups. There were no litters comprising stillborn pups only.

ORGAN WEIGHTS OF PARENTAL ANIMALS
No effects on organ weights were observed in male and female animals.

MACROSCOPIC EXAMINATION OF PARENTAL ANIMALS
At necropsy many animals treated with the test substance showed blue (or bluish/green) discoloration of the (contents of the) gastro-intestinal tract and in a few cases also at mesenteric lymph nodes (5/24 rats). This was obviously due to the color of the test substance (passive effect). One male of the low dose group, no 34, had a green nodule (2 cm) in the thoracic cavity, which appeared to be an abscess. This was clearly caused by faulty dosing (which means that the gavage needle accidently had penetrated the esophagus wall), resulting in a local abscess with blue coloured particles present in the interstitial tissue. In one female of the low-dose group, no 45, the vaginal aperture was missing, which accounted for the dilated uterus and vagina and the observation that this animal had not mated. The other gross changes were unremarkable.

MICROSCOPIC EXAMINATION OF PARENTAL ANIMALS
As a standard procedure at necropsy the intestines were flushed with formalin to enable proper preservation of the mucosa. Consequently the blue discoloured contents were flushed out for a large part and therefore not present in the slides. In several animals some intraluminal blue content was still visible in slides of the intestines. It was noted at the microscopic evaluation, but was omitted from the summarizing histopathology table because of its limited relevance. There was no clear evidence that the blue substance was able to pass the intestinal wall, because it was only found in the lumen of the intestinal tract but not in other organs or tissues. There were a few exceptions though: there were two cases of faulty dosing in the low dose group (animals 34 (see above) and 44 (with widespread blue particles in the lungs; this animal was found dead on day 17 of the study) and in the high dose group (animal 84 which had some blue particles in the surrounding adipose tissue of the mesenteric lymph nodes which could not be explained). Further, microscopic examination of the sampled organs and tissues did not reveal treatment related histopathological changes. The histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They were common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to treatment.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
LITTER SIZE AND SEX
The mean number of pups delivered per litter was comparable in all groups (12.6, 11.6, 13.3 and 11.8 in the control, low dose, mid dose and high dose groups, respectively). The mean percentage of male littermates at delivery was comparable in all groups (52.2%, 50.4%, 49.3% and 49.6% in the control, low dose, mid dose and high dose groups, respectively).

PUP OBSERVATIONS
Five female pups from one litter in the mid dose group were found to be cold and another female pup of this litter showed paralyzed legs. No treatment related clinical signs were observed during lactation.

PUP BODY WEIGHT
Mean pup body weight was comparable for males and females in all groups on days 0 and 4 of lactation.

VIABILITY INDEX
A total number of 113, 115, 146 and 141 pups were liveborn in the control, low dose, mid dose and high dose groups, respectively. One pup in the low dose group was stillborn and a total of 4, 3 and 9 pups in the control, low dose and mid dose group were missing and had died on day 4. No pups in the high dose were lost during day 0 and 4 of lactation. There were no litters showing complete litter loss. The viability indices on day 4 of lactation (96.5%, 97.4%, 93.8% and 100% for the control, low dose, mid dose and high dose groups, respectively) were comparable in all groups.

MACROSCOPIC OBSERVATIONS OF STILLBORN PUPS OR PUPS THAT DIED DURING LACTATION
Macroscopic examination of the stillborn pup in the low dose group (31-06) showed that the pup had no milk in the stomach.

Abnormalities:

not specified

Developmental effects observed:

not specified

A homogeneous distribution of the test item in the dose formulation was ensured by means of continuous stirring prior to dosing. The achieved concentrations were generally considered acceptable.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Please refer to the discussion of "Effects on fertility" (see above).

Justification for selection of Effect on developmental toxicity: via oral route:
The surrogate CAS number 75247-18-6 is used for a read-across to the target substance CAS number 28654-73-1.

Justification for classification or non-classification

Based on the results obtained from reproduction/developmental testing of the read across substance, the test substance is not considered to be subject for classification and labelling for toxicity to reproduction/development according to Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (CLP).